CN109982720A - Antibody drug conjugate - Google Patents

Abstract

The present invention relates to the antibody-drug conjugates comprising antibody, ferric iron and at least one drug molecule, and the pharmaceutical composition comprising the antibody-drug conjugates.The invention further relates to purposes of the antibody-drug conjugates in treatment disease such as cancer.

Description

Antibody Drug Conjugates

background

Conventional cancer chemotherapy is often accompanied by systemic toxicity to patients. Targeted therapeutic approaches seek to specifically interfere with molecular targets and pathways important for cancer cell proliferation. These targets are preferentially expressed intracellularly or on the surface of tumor cells. Thus, targeted therapy offers the potential to generate active agents that are selectively cytotoxic to tumor cells, while being less toxic to the host, resulting in a greater therapeutic index. One approach is to develop tyrosine kinase inhibitors that are misregulated and/or overexpressed in cancer cells. Another targeted therapy approach is the use of small molecules to selectively bind to the tumor cell surface to deliver cytotoxic compounds (eg, folate-vinca alkaloid conjugates).

Monoclonal antibodies against antigenic cancer cells offer an alternative tumor-selective therapy. However, many monoclonal antibodies do not have sufficient therapeutic activity on their own. Antibody-drug conjugates (ADCs) use antibodies to selectively deliver a potent cytotoxic compound to tumor cells, thereby improving the therapeutic index of chemotherapeutic agents. The two most prominent examples are the approved drug trastuzumab-emtansine (ado-trastuzumab emtansine) and brentuximab vedotin

However, current ADCs often experience insufficient ADC potency compared to the parent free drug. This may be attributed to insufficient antibody cleavage after ADC uptake by cancer cells and processing in endosomes/lysosomes. Current ADCs often require the use of specialized linkers that are not or only partially cleaved to the antibody during intracellular metabolism.

Another disadvantage of some ADCs is their poor stability in blood. The unstable linker releases the drug prematurely, resulting in loss of ADC activity and increased adverse side effects. On the other hand, once the ADC reaches the endosome, the highly stable linker prevents efficient release.

In addition, it is often difficult to obtain sufficiently high intracellular concentrations of cytotoxic compounds because the number of antigens ^on cancer cells is usually < 105. However, the intracellular concentration of the cytotoxic compound should preferably be significantly higher than ¹⁰⁵ .

WO 2013/103707 A1 discloses various ADCs comprising platinum cations.

Accordingly, there is a need for improved antibody drug conjugates that overcome one or more of the shortcomings of current therapies.

SUMMARY OF THE INVENTION

The inventors of the present application found that ADCs in which a drug is linked to an antibody via ferric iron are stable and selective, and can transport a large number of drug molecules into target cells. Furthermore, the intracellular release of the active agent is improved due to the reduction in iron cations only in the endosome. Undesirable drug release in the bloodstream is thereby prevented or at least substantially reduced.

Accordingly, the present invention relates to, but is not limited to, the embodiments defined in the following items [1] to [43]:

[1] An antibody-drug conjugate comprising an antibody, ferric iron bound to the antibody, and at least one drug molecule bound to ferric iron.

[2] The antibody-drug conjugate of item [1], wherein ferric iron is complexed with the antibody and the drug molecule.

[3] The antibody-drug conjugate of item [1] or [2], wherein ferric iron is bound to the antibody through a first linker.

[4] The antibody-drug conjugate of item [3], wherein the first linker comprises an iron complex group capable of binding ferric iron.

[5] The antibody-drug conjugate of item [3] or [4], wherein the first linker is covalently bound to the antibody.

[6] The antibody-drug conjugate of any one of the above items, wherein at least two drug molecules are bound to ferric iron.

[7] The antibody-drug conjugate of any one of the above items, wherein the average number of drug molecules per antibody in the conjugate is at least 5.

[8] The antibody-drug conjugate of any one of the above items, wherein the average number of drug molecules per antibody in the conjugate is at least 10.

[9] The antibody-drug conjugate of any one of the above items, wherein the average number of drug molecules per antibody in the conjugate is at least 15.

[10] The antibody-drug conjugate of any one of the above items, wherein the drug molecule is not released from the antibody-drug conjugate when the antibody-drug conjugate is in human blood.

[11] The antibody-drug conjugate of any one of the above items, wherein the drug molecule is released from the antibody-drug conjugate when the antibody-drug conjugate is in the endosomal compartment of a human cell.

[12] The antibody-drug conjugate of any one of the above items, wherein the drug molecule is selected from the group consisting of anti-cancer agents, anti-inflammatory agents and anti-infective agents.

[13] The antibody-drug conjugate of any one of the above items, wherein the drug molecule is an anticancer agent.

[14] The antibody-drug conjugate of item [13], wherein the anticancer agent is selected from the group consisting of microtubule structure formation inhibitors, meiosis inhibitors, RNA polymerase inhibitors, topoisomerase inhibitors, DNA intercalators , DNA alkylating agents and ribosome inhibitors.

[15] The antibody-drug conjugate of any one of the above items, wherein the antibody is capable of binding to an antigen expressed on tumor cells.

[16] The antibody-drug conjugate of any of the above items, wherein the drug molecule comprises an iron complexing group bound to ferric iron.

[17] The antibody-drug conjugate of any one of items [1] to [15], wherein the drug molecule is covalently linked to an iron complex group bound to ferric iron.

[18] The antibody-drug conjugate of item [16] or [17], wherein the iron complex group is selected from a pyridine derivative, a hydroxamic acid group, a catechol group, a carboxylic acid group and its acid.

[19] The antibody-drug conjugate of any one of the above items, which has the following structure:

Ab[-L1-Me(-L2-D) _n ] _m ,

in

Ab is an antibody,

L1 is the first connector,

Me is ferric iron,

L2 is the second linker or is absent,

D is the drug molecule,

m is a numerical value in the range 1 to 10, and

n is a numerical value ranging from 1 to 3.

[20] The antibody-drug conjugate of item [19], wherein m is a numerical value ranging from 2 to 6.

[21] The antibody-drug conjugate of item [19], wherein m is a numerical value ranging from 3 to 5.

[22] The antibody-drug conjugate of any one of items [19] to [21], wherein n is 2 or 3.

[23] The antibody-drug conjugate of any of the above items, wherein if the ferric iron is reduced, the drug molecule is released from the ferric iron.

[24] The antibody-drug conjugate of any of the above items, wherein the drug molecule is released from ferric iron at pH less than 5.

[25] The antibody-drug conjugate of any one of the above items, which is stable at pH 8.

[26] The antibody-drug conjugate of any one of the above items, which is stable at pH 7.

[27] The antibody-drug conjugate of any one of the above items, which is stable at pH 6.

[28] The antibody-drug conjugate of any one of the above items, which is stable at pH 5.

[29] A pharmaceutical composition comprising the antibody-drug conjugate of any one of the above items.

[30] The pharmaceutical composition of item [29], which further comprises a pharmaceutically acceptable excipient.

[31] The antibody-drug conjugate of any one of items [1] to [28], which is used as a therapeutic agent.

[32] The antibody-drug conjugate of any one of items [1] to [28] for use in the treatment of disorders.

[33] The antibody-drug conjugate of any one of items [1] to [28], which is for use in the treatment of cancer.

[34] The antibody-drug conjugate for the use of item [33], wherein the treatment comprises administering to a patient the antibody-drug conjugate of any one of items [1] to [28] and a drug different from the Anticancer agents of antibody-drug conjugates.

[35] Use of ferric iron for preventing or reducing the release of a drug molecule in blood from an antibody-drug conjugate, wherein the ferric iron is complexed with the antibody and the drug molecule to form the antibody-drug conjugate.

[36] The use of item [35], wherein the drug molecule is released from the antibody-drug conjugate in the endosomal compartment of the cell.

[37] The use of item [35] or [36], wherein the antibody drug-molecule conjugate is an antibody-drug conjugate as defined in any one of items [1] to [28].

[38] Use of the antibody-drug conjugate of any one of items [1] to [28] for preventing or reducing the release of a drug molecule from the antibody-drug conjugate in blood.

[39] A method of preventing or reducing the release of drug molecules from an antibody-drug conjugate in blood, the method comprising complexing ferric iron with an antibody and a drug to form an antibody-drug conjugate.

[40] The method of item [39], wherein the antibody-drug conjugate is an antibody-drug conjugate as defined in any one of items [1] to [28].

[41] Use of ferric iron in linking a drug molecule to an antibody.

[42] The use of item [41], wherein an antibody-drug conjugate is obtained by the linkage.

[43] The use of item [41] or [42], wherein the antibody-drug conjugate is an antibody-drug conjugate as defined in any one of items [1] to [28].

Brief Description of Drawings

Figure 1. A new concept for ADCs: Fe-complexes as pH/Fe-reductase labile linkers. Cytotoxic compounds are conjugated to iron-complexed ligands. This complex is then conjugated to an antibody that has its natural receptor on cancer cells. Upon antibody-antigen binding, the entire ADC is taken up by endocytosis and the complex disintegrates in vivo due to low pH and/or Fc reductase activity, thereby releasing the drug. Further action such as peptidases may be required to release the active entity.

Figure 2. Iron may be complexed by cytotoxic drugs themselves. In this example, a histone deacetylase inhibitor such as SAHA (Vorinostat) is used to complex iron. This is an example of trace-free drug release from an iron-based ADC. The hydroxamic acid functional group achieves iron complexation through the drug itself without any modification of the molecule.

Figure 3. The very potent drug doxorubicin can also be used to complex iron after some modification of the amine functional group. A further mode of action is shown in Figure 1.

Figure 4. The upper graph shows an analytical LC-run of complex A at pH 5.0, 37°C for 16 hours. The lower panel shows an analytical LC-run of complex A at pH 8.0, 37°C over 16 hours (see Example 1).

Figure 5. Overlay of analytical HPLC chromatograms of the ligands used in complexes A and B, their respective iron complexes, and an additional sample of preformed complex A with free ligand B showing no intermediate complex (Example 2).

Figure 6. Overlay of analytical HPLC chromatograms of the free ligands (ST116), their respective complexes, and complexes resulting in hydrogen reduction of the respective free ligands B (Example 3).

Figure 7. Conjugation of iron-complexing moieties to AB to produce product AB-1 followed by iron complexation (AB-2) with two auxiliary ligands (Example 4).

Figure 8. Top panel: Fluorescence measurements of conjugated/complexed AB-2 and unconjugated AB. Bottom panel: difference in fluorescence of complexed and non-complexed conjugates (Example 4).

Figure 9. Fluorescence spectra of AB-2 (purple) and its reduced derivative AB-1 (green), where the reduced complex reduces fluorescence by -20% (Example 4).

Figure 10. Fluorescence of ornithine N-hydroxamic acid coupled to fluorescein amine (ST102) is quenched by Fe3 ⁺ , but regains fluorescence upon reduction of ^{Fe3 +} to Fe2+ (Example 5) .

Detailed Description

In a first aspect, the present invention relates to an antibody-drug conjugate comprising an antibody, at least one ferric iron bound to the antibody, and at least one drug molecule bound to the ferric iron.

Antibody

As used herein, the term "antibody" refers to an immunoglobulin (Ig), which is defined as a protein belonging to the IgG, IgM, IgE, IgA, or IgD class (or any subclass thereof), or a functional fragment thereof. In the context of the present invention, a "functional fragment" of an immunoglobulin is defined as an antigen-binding fragment or other derivative of a parent immunoglobulin that substantially maintains the antigen-binding activity of such parent immunoglobulin. Functional fragments are antibodies in the sense of the present invention, even if they have a lower affinity for the antigen than the parent immunoglobulin has for the antigen. "Functional fragments" of the present invention include F(ab') ₂ fragments, Fab fragments, scFvs, dsFvs, dimeric, trimeric, tetrameric and Fc fusion proteins. F(ab') ₂ or Fab can be engineered to minimize or completely remove the intermolecular disulfide interactions that occur between the CH1 and CL domains. Antibodies of the invention may be part of a bifunctional construct or a multifunctional construct. Antibodies of the present invention include, but are not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-genotypic antibodies.

Preferably, the antibodies of the present invention are monoclonal antibodies.

The antibodies of the ADCs of the present invention preferably specifically bind to antigens expressed on the surface of cancer cells.

drug molecule

The term "drug molecule" or "payload" as used herein refers to a therapeutic or diagnostic agent. Preferred drug molecules include anticancer, anti-inflammatory, and anti-infective agents (eg, antifungal, antibacterial, antiparasitic, antiviral).

Preferably, the drug molecule of the present invention is an anticancer agent. Suitable anticancer agents include, but are not limited to, alkylating agents, antimetabolites, spindle poisons plant alkaloids, cytotoxic/antineoplastic antibiotics, topoisomerase inhibitors, photosensitizers, and kinase inhibitors.

Also included in the definition of "anticancer agent" are: (i) antihormonal agents, such as antiestrogens and selective estrogen receptor modulators, for modulating or inhibiting hormonal effects on tumors; (ii) inhibiting aromatase an aromatase inhibitor that modulates estrogen production in the adrenal gland; (iii) an antiandrogen; (iv) a protein kinase inhibitor; (v) a lipid kinase inhibitor; (vi) an antisense oligonucleotide, Especially those antisense oligonucleotides that inhibit gene expression in signaling pathways involved in abnormal cell proliferation; (vii) ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines; A construct 1 inhibitor; (ix) an anti-angiogenic agent; and any of the above pharmaceutically acceptable salts, acids, solvates and derivatives.

The most preferred anticancer agents in the present invention are those which are removed almost without trace after disintegration of the iron-ligand complex. This means that compounds with high binding affinity to their targets need not be or are slightly modified to complex with iron. Most notable in this example are compounds such as the HDAc inhibitor ST-3595 (see Figure 2). However, different compounds may also be well suited for complexation with ferric iron while being able to disintegrate upon iron reduction/ligand protonation.

ferric iron

The term "ferric iron" as used herein refers to iron cations with oxidation number +3, also known as iron(III), Fe(III) or Fe3 ⁺ .

In general, pharmaceutically acceptable transition metal cations capable of forming complexes with other compounds include, for example, cations of platinum, ruthenium, iridium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, and zinc. However, the transition metal cation in the ADC of the present invention is ferric iron.

conjugate

Typically, ferric iron is bound indirectly to the antibody through a first linker.

As used herein, the term "linker" refers to a chemical moiety that attaches a molecule or atom to a chemical compound such as an antibody or drug molecule. Typically, linkers comprise chains of atoms connected by chemical bonds.

The first linker links the ferric iron to the antibody.

One end of the first linker is preferably covalently linked to the antibody. The antibody-reactive end of the first linker is usually the site capable of conjugation to the antibody through a cysteine thiol or lysine amine group on the antibody, and is therefore usually a thiol-reactive group such as a double bond (as in maleimide) or leaving groups such as chlorine, bromine or iodine, or R-sulfanyl, or amine reactive groups such as carboxyl. When the term "linker" is used to describe the linker in conjugated form, one reactive end will be absent (eg, the leaving group of a thiol-reactive group) or incomplete (eg, only the carbonyl group of the carboxylic acid will be present) , because a bond is formed between the linker and the antibody. Different linker types are described in McCombs et al. (2015) The AAPS Journal, Vol. 17, No. 2, pp. 339-351, and Lu et al. (2016) Int. J. Mol. Sci. 17, 561 (doi: 10.3390/ ijms17040561), the contents of which are incorporated herein by reference.

Linkers can contain a variety of chemical groups including, but not limited to, optionally substituted divalent groups such as alkylene, arylene, heteroarylene; moieties such as: -(CR ₂ ) _n O(CR ₂ ) _n- , repeating units of alkoxy groups (eg, polyvinyloxy, PEG, polymethyleneoxy) and repeating units of alkylamino groups (eg, polyvinylamino); and di-esters and amides, including succinic acid esters, succinamide, diglycolate, malonate, and caproamide; wherein each R is independently H, _C1 - _C18 alkyl, _{C6 - C20} aryl, C3- _C14 heterocycle , a protecting group or a prodrug moiety, and n is an integer from 1 to 10.

In particular embodiments, the linker may be a peptide comprising one or more amino acid units. Examples of amino acid linkers include dipeptides, tripeptides, tetrapeptides or pentapeptides. Amino acid linker moieties may or may not be designed and optimized for their selectivity for enzymatic cleavage by a particular enzyme.

The second end of the first linker preferably contains or consists of an iron complexing group capable of binding the ferric iron of the ADC. The iron complexing group is preferably a chelating group. More preferably, the iron complexing group is a chelating group of a siderophore. In a preferred embodiment, the iron complexing group is a catechol group, a carboxylic acid group, or a hydroxamic acid group, as shown in the following formulae (I) to (III), respectively, wherein R is a group comprising a first the remainder of the linker at the end.

As used herein, the terms catechol group, carboxylic acid group, and hydroxamic acid group include their corresponding acid and deprotonated forms.

A preferred first linker for the ADC of the present invention is a compound of formula (I) wherein R is -( _CH2 ) _p - ^CH (NHR2)-(C=O)... and p is 0 to 6 An integer of , R ² is formyl, acetyl, peptidyl, or C ₁ -C ₂₀ hydrocarbyl, and where the dotted line represents the binding site with the antibody. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

A particular first linker of the ADC of the present invention is a compound of formula (I) wherein R is -( _CH2 ) _p -CH( _NH2 )-(C=O)..., p is 0 to 6 Integer numbers, and dotted lines indicate binding sites with antibodies. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

Another preferred first linker for the ADC of the present invention is a compound of formula (I) wherein R is -C=O-NH-( _CH2 ) _p - ^CH (NHR2)-(C=O)... ·, p is an integer from 0 to 6, R ² is formyl, acetyl, peptidyl or C ₁ -C ₂₀ hydrocarbyl, and wherein the dotted line represents the binding site with the antibody. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

Another particular first linker of the ADC of the present invention is a compound of formula (I) wherein R is -C=O-NH-( _CH2 ) _p -CH( _NH2 )-(C=O)... ·, p is an integer from 0 to 6, and the dotted line represents the binding site with the antibody. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

A preferred first linker for the ADC of the present invention is a compound of formula (II) wherein R is -( _CH2 ) _p - ^CH (NHR2)-(C=O)..., p is 0 to 6 Integer, R2 is formyl, acetyl, peptidyl, or _C1 ^- _C20 hydrocarbyl, and where the dotted line represents the binding site with the antibody. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

A particular first linker of the ADC of the present invention is a compound of formula (II) wherein R is -( _CH2 ) _p -CH( _NH2 )-(C=O)..., p is 0 to 6 Integer numbers, and dotted lines indicate binding sites with antibodies. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

Another preferred first linker for the ADC of the present invention is a compound of formula (II) wherein R is -C=O-NH-( _CH2 ) _p - ^CH (NHR2)-(C=O)... ·, p is an integer from 0 to 6, R ² is formyl, acetyl, peptidyl or C ₁ -C ₂₀ hydrocarbyl, and the dotted line represents the binding site with the antibody. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

Another particular first linker of the ADC of the present invention is a compound of formula (II) wherein R is -C=O-NH-( _CH2 ) _p -CH( _NH2 )-(C=O)... ·, p is an integer from 0 to 6, and the dotted line represents the binding site with the antibody. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

A preferred first linker for the ADC of the present invention is a compound of formula (III) wherein R is -( _CH2 ) _p - ^CH (NHR2)-(C=O)..., p is 0 to 6 Integer, R ² is formyl, acetyl, peptidyl, or C ₁ -C ₂₀ hydrocarbyl, and the dotted line represents the binding site with the antibody. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

A particular first linker of the ADC of the present invention is a compound of formula (III) wherein R is -( _CH2 ) _p -CH( _NH2 )-(C=O)..., p is 0 to 6 Integer numbers, and dotted lines indicate binding sites with antibodies. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

Another preferred first linker for the ADC of the present invention is a compound of formula (III) wherein R is -C=O-NH-( _CH2 ) _p - ^CH (NHR2)-(C=O)... ·, p is an integer from 0 to 6, R ² is formyl, acetyl, peptidyl or C ₁ -C ₂₀ hydrocarbyl, and the dotted line represents the binding site with the antibody. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

A particular first linker of the ADC of the present invention is a compound of formula (III) wherein R is -C=O-NH-( _CH2 ) _p -CH(NH2)-(C=O)...,p is an integer from 0 to 6, and the dotted line represents the binding site with the antibody. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

In other preferred embodiments of the present invention, the iron complexing group is a pyridine derivative capable of complexing ferric iron as defined above. Suitable pyridine derivatives are described, for example, in "The Chemistry of Heterocyclic Compounds, pyridine Metal Complexes", ed. Desmond J. Brown, John Wiley & Sons, 2009 (ISBN-13:9780470239728), the disclosure of which is incorporated herein in its entirety. The embodiments described above in connection with formula (I), (II) or (III) apply mutatis mutandis to the other chelating agents described in this reference.

Other suitable iron complexing groups are described in "Chelating Agents and Metal Chelates", ed. FDwyer, 2012 (ISBN-13:9780323146418), the disclosure of which is incorporated herein in its entirety. The embodiments described above in connection with formula (I), (II) or (III) apply mutatis mutandis to the other chelating agents described in this reference.

The average number of first linkers per antibody in the ADCs of the invention (ie, the molar "first linker-to-antibody ratio") is generally at least 1, preferably at least 2, more preferably at least 5, more preferably at least 8, more preferably At least 10, most preferably >10.

The molar "first linker-antibody ratio" in the ADCs of the invention is generally 1 to 50, preferably 2 to 40, more preferably 5 to 35, more preferably 8 to 30, most preferably 10 to 20, eg 11 to 20, 12 to 20, 13 to 20, 14 to 20 or 15 to 20. In other embodiments, the molar "first linker-antibody ratio" is 10 to 19, or 11-18, or 12 to 17, or 13 to 16, such as 13, 14, 15 or 16.

The molar "first linker-antibody ratio" in the ADC of the invention is preferably >10.

The ADCs of the invention may comprise one or more ferric iron per antibody molecule. The amount of ferric iron per antibody molecule (ie the molar "ferric-antibody ratio") is preferably equal to the amount of first linker conjugated to the antibody, in which case the first linker is capable of complexing only one Trivalent iron. Depending on the chemical design, the first linker may be capable of complexing more than one ferric iron, eg 2 or 3 ions (see formula below).

The average number of drug molecules per antibody in an ADC of the invention (ie, the molar "drug-to-antibody ratio") is typically twice the number of first linkers conjugated to the antibody. So usually 2 to 30 or 4 to 20. In the example in the above formula, one first linker is used to complex 3 ferric iron, where 2*3=6 concomitant drug molecules. In the ADC of the present invention, the drug molecule is directly or indirectly bound to ferric iron. Conjugation is preferably achieved through an iron complexing group as defined above for the first linker.

If the drug molecule binds directly to ferric iron, the drug molecule itself is capable of binding to ferric iron. According to this embodiment, the drug molecule preferably comprises an iron complexing group. The iron complexing group may be the same or different as defined above for the first linker. Examples of drug molecules capable of binding ferric iron include the following HDAC inhibitors:

It can be seen that the drug molecule contains a hydroxamic acid group capable of binding ferric iron. This is also exemplified in Figure 2. Also shown on the left side of Figure 3 is an embodiment where the drug molecule (doxorubicin) is modified to allow binding to ferric iron. Since the released modified drug molecule is active, this is still considered a "direct binding" of the drug molecule to ferric iron.

If the drug molecule binds ferric iron indirectly, the drug molecule is attached to a second linker comprising a chemical group capable of binding ferric iron. The chemical group capable of binding ferric iron is usually a metal complexing group capable of binding ferric iron. The preferred iron complexing groups described above in conjunction with the first linker are also preferred iron complexing groups for the second linker. That is, one end of the second linker preferably contains or consists of a hydroxamic acid group, a catechol group or a carboxylic acid group. The other end of the second linker is preferably covalently linked to the drug molecule. This embodiment is illustrated in Figure 1, where the drug molecule is referred to as the "payload". Also shown on the right side of Figure 3 is an embodiment in which doxorubicin is attached to a linker comprising an iron complexing group to allow binding to ferric iron.

Preferred second linkers for the ADCs of the invention are compounds of formula (I) wherein R is -( _CH2 ) _p - ^CH (NHR2)-(C=O)... and p is an integer from 0 to 6 , R ² is formyl, acetyl, peptidyl or C ₁ -C ₂₀ hydrocarbyl, and where the dotted line represents the binding site with the drug molecule. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

A particular second linker of the ADC of the present invention is a compound of formula (I) wherein R is -( _CH2 ) _p -CH( _NH2 )-(C=O)... and p is an integer from 0 to 6 , and the dotted line indicates the binding site with the drug molecule. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

Another preferred second linker of the ADC of the present invention is a compound of formula (I), wherein R is -C=O-NH-( _CH2 ) _p - ^CH (NHR2)-(C=O)... , p is an integer from 0 to 6, R ² is formyl, acetyl, peptidyl or C ₁ -C ₂₀ hydrocarbyl, and the dotted line represents the binding site with the drug molecule. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

Another particular second linker of the ADC of the invention is a compound of formula (I) wherein R is -C=O-NH-( _CH2 ) _p -CH( _NH2 )-(C=O)... , p is an integer from 0 to 6, and the dotted line represents the binding site with the drug molecule. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

A preferred second linker for the ADC of the present invention is a compound of formula (II), wherein R is -( _CH2 ) _p - ^CH (NHR2)-(C=O)... and p is an integer from 0 to 6 , R ² is formyl, acetyl, peptidyl or C ₁ -C ₂₀ hydrocarbyl, and where the dotted line represents the binding site with the drug molecule. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

A particular second linker of the ADC of the present invention is a compound of formula (II) wherein R is -( _CH2 ) _p -CH( _NH2 )-(C=O)... and p is an integer from 0 to 6 , and the dotted line indicates the binding site with the drug molecule. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

Another preferred second linker of the ADC of the present invention is a compound of formula (II), wherein R is -C=O-NH-( _CH2 ) _p - ^CH (NHR2)-(C=O)... , p is an integer from 0 to 6, R ² is formyl, acetyl, peptidyl or C ₁ -C ₂₀ hydrocarbyl, and the dotted line represents the binding site with the drug molecule. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

Another particular second linker of the ADC of the invention is a compound of formula (II) wherein R is -C=O-NH-( _CH2 ) _p -CH( _NH2 )-(C=O)... , p is an integer from 0 to 6, and the dotted line represents the binding site with the drug molecule. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

A preferred second linker for the ADC of the present invention is a compound of formula (III), wherein R is -( _CH2 ) _p - ^CH (NHR2)-(C=O)... and p is an integer from 0 to 6 , R ² is formyl, acetyl, peptidyl or C ₁ -C ₂₀ hydrocarbyl, and where the dotted line represents the binding site with the drug molecule. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

A particular second linker of the ADC of the present invention is a compound of formula (III) wherein R is -( _CH2 ) _p -CH( _NH2 )-(C=O)... and p is an integer from 0 to 6 , and the dotted line indicates the binding site with the drug molecule. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

Another preferred second linker of the ADC of the present invention is a compound of formula (III), wherein R is -C=O-NH-( _CH2 ) _p - ^CH (NHR2)-(C=O)... , p is an integer from 0 to 6, R ² is formyl, acetyl, peptidyl or C ₁ -C ₂₀ hydrocarbyl, and the dotted line represents the binding site with the drug molecule. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

Another particular second linker of the ADC of the present invention is a compound of formula (III), wherein R is -C=O-NH-( _CH2 ) _p -CH( _NH2 )-(C=O)... , p is an integer from 0 to 6, and the dotted line represents the binding site with the drug molecule. Preferably, p is an integer from 2 to 5, more preferably p is 3 or 4, most preferably p is 3.

In one embodiment, the second linker of the ADC of the invention is a non-cleavable linker. Examples of non-cleavable linkers are maleimide-alkane linkers (eg, maleimido-hexanoyl linkers) and maleimide-cyclohexane linkers.

In another embodiment, the second linker of the ADC of the invention is a cleavable linker. Cleavable linkers include chemically labile linkers and enzymatically cleavable linkers. The chemically labile linker can be an acid-cleavable linker or a reducible linker. The acid-cleavable linker may contain a hydrazone group. The reducible linker may contain a disulfide group. Enzymatically cleavable linkers typically contain chemical groups that can be cleaved or degraded by one or more lysosomal enzymes. Suitable groups include a valine-citrulline dipeptide group, a phenylalanine-lysine dipeptide group, and a beta-glucuronide group (which can be cleaved by beta-glucuronidase).

In a particular embodiment of the invention, the first linker is a non-cleavable linker and the second linker is a cleavable linker. In another particular embodiment of the present invention, the first linker is a non-cleavable linker and the second linker is a non-cleavable linker. In another particular embodiment of the present invention, the first linker is a cleavable linker and the second linker is a cleavable linker. In another particular embodiment of the present invention, the first linker is a cleavable linker and the second linker is a non-cleavable linker.

Drug molecules are released from the ADC upon internalization of the ADC by target cells, preferably cancer cells. Without wishing to be bound by theory, it is believed that release is enhanced by low pH and by reduction of ferric iron, eg, by the enzyme ferroreductase. Due to lower pH and increased presence of ferroreductase in endosomes, Fe ³⁺ -based ADC complexes should be broken down in endosomes, thereby enhancing the release or diffusion of smaller ligands into the intracellular space.

In one embodiment, the drug molecule is released from the ADC of the invention when the ADC is located in the endosomal compartment of a cell, preferably a mammalian cell, most preferably a human cell.

Preferably, at least 25%, more preferably at least 50%, more preferably at least 75%, most preferably at least 95% of the drug molecules are released from the ADC upon reduction of ferric iron to ferrous iron, as described in the Examples Measured by the assay described in 3.

In another embodiment, the ADCs of the invention are stable at pH 8, as determined in the assay described in Example 1. In another embodiment, the ADCs of the invention are stable at pH 7, as determined in the assay described in Example 1. In another embodiment, the ADCs of the invention are stable at pH 6, as determined in the assay described in Example 1. In another embodiment, the ADCs of the invention are stable at pH 5. As determined in the assay described in Example 1. "Stable" in the sense of this assay means that incubation of the complex under the conditions of Example 1 (pH 5 and pH 8) does not result in the release of free ligand, as in the second diagram according to Figure 4 peak observed.

Preferably, the drug molecule is not released from the ADC of the invention when the antibody is in blood, preferably in human blood. In other embodiments, the drug molecule is not released from the ADC of the invention when the antibody is in plasma, preferably plasma from a vertebrate, more preferably human plasma.

In another embodiment, the ADCs of the invention exhibit substantially no ligand exchange, as determined in the assay described in Example 2.

Another aspect of the present invention is an ADC having the following structure:

Ab[-L1-Me(-L2-D) _n ] _m ,

in

Ab is an antibody,

L1 is the first connector,

Me is ferric iron,

L2 is the second linker or absent,

D is the drug molecule,

m is 1 to 10, and

n is 1 to 3.

The preferred embodiments of the antibody, first and second linker, ferric iron and drug molecule described above apply mutatis mutandis to this aspect of the invention. The parameter m is preferably 2 to 6, or 3 to 5, eg 3, 4 or 5. The parameter n is preferably 2 or 3, most preferably 2. In particular embodiments, m and/or n are integers.

In a preferred embodiment, n is 2 and m is 2.

In another preferred embodiment, n is 2 and m is 3.

In another preferred embodiment, n is 2 and m is 4.

In another preferred embodiment, n is 2 and m is 5.

In another preferred embodiment, n is 2 and m is 6.

In another preferred embodiment, n is 2 and m is 7.

In another preferred embodiment, n is 2 and m is 8.

In another preferred embodiment, n is 2 and m is 9.

In another preferred embodiment, n is 2 and m is 10.

In another preferred embodiment, n is 3 and m is 2.

In another preferred embodiment, n is 3 and m is 4.

In another preferred embodiment, n is 3 and m is 5.

In another preferred embodiment, n is 3 and m is 6.

In another preferred embodiment, n is 3 and m is 7.

In another preferred embodiment, n is 3 and m is 8.

In another preferred embodiment, n is 3 and m is 9.

In another preferred embodiment, n is 3 and m is 10.

Preparation of ADCs

Another aspect of the invention is a method of making the ADC of the invention. The linker can be synthesized by a method including steps known per se.

ADCs of the present invention can be prepared as follows:

In the present invention, as described in Example 5, an iron-complexing moiety is covalently linked to an antigen-recognition structure of interest (eg, an antibody). The most straightforward method is via the succinimide-activated conjugation used in Example 5. However, various conjugation strategies (eg maleimide and click chemistry methods) are described in the literature. After conjugation of the first linker, the coupling product is purified and subsequently separately added to a) two equivalents of the payload (this is the iron complexing compound or any compound attached to the iron complexing moiety) and b) one equivalent of the iron salt (In the case of Example ₅ , this was FeCl3) were incubated together. The resulting product was then purified again using a size exclusion column.

treat

In one embodiment, the present invention provides a method of treating or preventing a disease comprising administering an ADC of the present invention to a patient, preferably a human patient. In certain embodiments, the disease to be treated or prevented is cancer.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is often characterized by unregulated cell growth. A "tumor" includes one or more cancer cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (eg, epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, Gastric cancer includes gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular tumor, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer Or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, and head and neck cancer.

For prophylaxis or treatment of disease, the appropriate dose of ADC will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the molecule is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to antibody response, and the judgment of the attending physician. The molecule is suitable for administration to a patient at one time or in a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (eg, 0.1-20 mg/kg) of the molecule is an initial candidate dose for administration to a patient, eg, by one or more separate administrations, or by Continuous infusion. A typical daily dose may range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. Exemplary doses of ADC administered to a patient range from about 0.1 to about 10 mg/kg of the patient's body weight.

For repeated administration over several days or longer, depending on the condition, treatment is continued until the desired suppression of disease symptoms occurs. An exemplary dosing regimen includes administration of an initial loading dose of about 4 mg/kg, followed by administration of a weekly maintenance dose of ADC of about 2 mg/kg. Other dosage regimens may be useful. The progress of this therapy can be readily monitored by conventional techniques and assays.

combination therapy

Antibody-drug conjugates (ADCs) can be combined with a second compound having anticancer properties as combination therapy in a pharmaceutical combination formulation or dosing regimen. The second compound of the pharmaceutical combination formulation or dosing regimen preferably has complementary activities to the ADCs of the combination such that they do not adversely affect each other.

The second compound can be a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitor, an antihormonal agent, an aromatase inhibitor, a protein kinase inhibitor, a lipid kinase inhibitor, an antiandrogen, an antisense oligonucleotide Acids, ribozymes, gene therapy vaccines, anti-angiogenic and/or cardioprotective agents. These molecules are suitably present in combination in amounts effective for the intended purpose. Pharmaceutical compositions containing ADCs may also have a therapeutically effective amount of a chemotherapeutic agent, such as a tubulin formation inhibitor, a topoisomerase inhibitor, or a DNA binding agent.

Other treatment regimens can be combined with the administration of anticancer agents identified according to the present invention. Combination therapy can be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations. Combination administration includes co-administration using separate formulations or a single pharmaceutical formulation, as well as sequential administration in either order, wherein there is a time period during which both (or all) active agents simultaneously exert their biological activities.

Combination therapy may provide and prove to be "synergistic", that is, the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by the compounds used alone. A synergistic effect may be obtained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined unit dose formulation; (2) delivered alternately or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect can be obtained when the compounds are administered or delivered sequentially, eg, using different injections in separate syringes. Typically, during alternation therapy, effective doses of each active ingredient are administered sequentially, ie, consecutively, while in combination therapy, effective doses of two or more active ingredients are administered together.

Also within the scope of the present invention are the in vivo metabolites of the ADC compounds described herein to the extent that such products are new and non-obvious compared to the prior art. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, enzymatic cleavage, and the like, of the administered compound. Accordingly, the present invention includes novel and non-obvious compounds produced by a method comprising contacting a compound of the present invention with a mammal for a period of time sufficient to produce a metabolite thereof.

Metabolites include in vivo cleavage products of ADCs, where cleavage of any bonds occurs that links the drug moiety to the antibody. Thus, metabolic cleavage can yield naked antibodies or antibody fragments. Antibody metabolites can be attached to part or all of the linker. Metabolic cleavage can also result in the production of drug moieties or portions thereof. The drug moiety metabolite may be attached to part or all of the linker.

Administration of Antibody-Drug Conjugate Pharmaceutical Formulations

Therapeutic ADCs can be administered by any route appropriate to the condition to be treated. ADCs are typically administered parenterally, ie by infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal, bolus, intratumoral injection or epidural administration (Shire et al. (2004) J. Pharm.Sciences 93(6):1390-1402). Pharmaceutical formulations of therapeutic antibody-drug conjugates (ADCs) for parenteral administration are typically prepared with pharmaceutically acceptable parenteral carriers and are in unit dose injectable form. Antibody-drug conjugates (ADCs) of desired purity are optionally mixed with pharmaceutically acceptable diluents, carriers, excipients or stabilizers in the form of lyophilized formulations or aqueous solutions (Remington's Pharmaceutical Sciences (1980) p. 16th edition, Osol, A. Ed.).

Acceptable parenteral vehicles, diluents, carriers, excipients and stabilizers are nontoxic to recipients at the dosages and concentrations used, and include buffers such as phosphates, citrates and other organic acids; antioxidants , including ascorbic acid and methionine; preservatives (e.g. octadecyldimethylbenzylammonium chloride; hexamethylene bisammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol) ; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine Acids, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; Salt-forming counterions, eg, sodium; metal complexes (eg, zinc-protein complexes); and/or nonionic surfactants, eg, TWEEN ^™ , PLURONICS ^™ , or polyethylene glycol (PEG). For example, lyophilized anti-ErbB2 antibody preparations are described in WO 97/0480.

Pharmaceutical formulations of therapeutic antibody-drug conjugates (ADCs) may contain amounts of unreacted drug moieties (D), antibody-linker intermediates (Ab-L) and/or drug-linker intermediates (DL) ) as a result of incomplete purification and separation of excess reagents, impurities and by-products during the preparation of ADCs or time/temperature hydrolysis or degradation when storing large quantities of ADCs or formulated ADC compositions. For example, formulations of ADCs may contain detectable amounts of free drug D. Alternatively, or in addition, it may also contain a detectable amount of the drug-linker intermediate D-L. Alternatively, or in addition, it may also contain a detectable amount of the antibody Ab. Exemplary formulations may contain up to 10% molar equivalents of free drug.

Active pharmaceutical ingredients can also be entrapped in microcapsules, eg, prepared by coacervation techniques or by interfacial polymerization, eg, in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, respectively). ) or hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules in macroemulsion. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Ed. (1980).

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing ADCs in the form of shaped articles, eg, films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (eg, poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (US Pat. No. 3,773,919), L-Valley Copolymers of amino acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT ^™ (consisting of lactic acid-glycolic acid copolymers and Injectable microspheres composed of leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.

Formulations for in vivo administration must be sterile, which can be readily accomplished by filtration through sterile filtration membranes.

Formulations include those suitable for the aforementioned routes of administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations are generally found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, pa.). These methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately bringing into association the active ingredient with a liquid carrier or a fine powder of a solid carrier, or both, and then, if desired, shaping the product.

Aqueous suspensions contain the active substance (ADC) in admixture with excipients suitable for the manufacture of aqueous suspensions. These excipients include suspending agents such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, Gum tragacanth and acacia, and dispersing or wetting agents such as naturally occurring phospholipids (eg, lecithin), condensation products of alkylene oxides with fatty acids (eg, polyoxyethylene stearate), ethylene oxide Condensation products with long-chain aliphatic alcohols (eg, heptadecylethoxycetyl alcohol), condensation products of ethylene oxide with partial esters and hexitol anhydrides derived from fatty acids (eg, polyoxyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and a or various sweeteners such as sucrose or saccharin.

Pharmaceutical compositions of ADCs can be in the form of sterile injectable preparations, such as sterile injectable aqueous or oily suspensions. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, such as a solution or preparation in 1,3-butanediol Freeze-dried powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables.

The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, an aqueous solution for intravenous infusion may contain from about 3 to 500 μg of active ingredient per milliliter of solution so that a suitable volume can be infused at a rate of about 30 mL/hour. Subcutaneous (bolus) administration can be performed with a total volume of about 1.5 mL or less and a concentration of about 100 mg ADC/mL. For ADCs requiring frequent and chronic administration, a subcutaneous route may be employed, such as by prefilled syringe or auto-injector device technology.

As a general recommendation, the initial pharmaceutically effective amount of ADC administered per dose will range from about 0.01 to 100 mg/kg, ie about 0.1 to 20 mg/kg of patient body weight per day, with a typical initial range for the compound used being 0.3 to 15 mg/kg/day sky. For example, a human patient may initially be dosed with about 1.5 mg ADC per kg of patient body weight. The dose can be escalated to the maximum tolerated dose (MTD). The dosing regimen can be about every 3 weeks, although the regimen can be more or less frequent depending on the condition or response diagnosed. The dose can be further adjusted to or below the MTD during treatment, which can be safely administered for multiple cycles, eg, about 4 or more.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatic agents and solutes to render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous non-aqueous Bacterial suspensions, which may contain suspending agents and thickening agents.

Although oral administration of protein therapeutics is often disadvantageous due to poor bioavailability due to limited absorption, hydrolysis or denaturation in the intestinal tract, ADC formulations suitable for oral administration can be prepared in discrete units such as capsules, cachets or tablets agents, each of which contains a predetermined amount of ADC.

The formulations can be packaged in unit-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, such as water, for injection immediately before use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the type previously described. Exemplary unit dosage formulations contain a daily dose or unit daily sub-dose or an appropriate fraction thereof of the active ingredient.

The present invention further provides a veterinary composition comprising at least one active ingredient as defined above and a veterinary carrier. Veterinary carriers are materials which can be used to administer the compositions and can be solid, liquid or gaseous materials which are inert or acceptable in the veterinary art and which are compatible with the active ingredient. These veterinary compositions can be administered parenterally, orally or by any other desired route.

A further aspect of the invention is the use of an ADC as described herein for preventing or reducing the release of drug molecules from the ADC in blood or plasma, preferably human blood or plasma. Another aspect of the invention is the use of ferric iron to prevent or reduce the release of drug molecules from antibody-drug conjugates in blood or plasma, preferably human blood or plasma, wherein ferric iron is complexed by the antibody and drug molecule to Antibody-drug conjugates are formed. Another aspect of the invention is a method of preventing or reducing the release of a drug molecule from an antibody-drug conjugate in blood, the method comprising complexing ferric iron with the antibody and the drug molecule to form the antibody-drug conjugate. Preferred embodiments of the uses and methods of the invention correspond to those of the ADC of the invention as described above.

Example

Example 1

To assess the stability of the complexes we envision for the above linkers, a number of experiments were performed. To this end, two model ligands (GVFe35 and ST088) were synthesized (see Scheme 1).

Scheme 1. Synthesis of two different hydroxamic acid compounds: GVFe35 and ST088.

Using these two ligands, the following two model complexes _were prepared by adding FeCl3 solution.

This formula shows the structure of the iron complex formed using GVFe35 (free carbonic acid, complex A) and ST088 (sec-butylamine coupled, complex B).

Complex stability was assessed at different pH over time, then complex A was incubated at pH 5.0 or pH 8.0 at 37°C. Final degradation of the complex was monitored by analytical HPLC at defined time intervals and after 16 hours. The HPLC chromatogram is shown in Figure 4. Subsequently, after 16 h, only modest degradation was observed in both cases, indicating that the complex is stable under these conditions.

Example 2

To check whether ligand exchange occurred (ie ligand dissociation and renewed complexation), additional experiments were performed whereby complexes A and B were mixed and incubated at 37°C, pH 7.0 for 16 hours. In the case of dissociation and subsequent complexation, intermediate complexes can be expected in which one or both ligands of complex B will complex iron with one ligand derived from complex A, and vice versa.

The complexes and mixtures were examined by analytical HPLC (see Figure 5). This complex has a longer retention time than complex A due to the more apolar sec-butylamide functionality in ST088 (for complex B). Therefore, in the case of ligand exchange, peaks with intermediate retention times will start to appear between the two complex peaks. Since no intermediate peaks were observed, this suggested that no ligand exchange had occurred for the complexes examined, indicating a high degree of stability.

Example 3

To further confirm this result and examine the effect of iron oxidation state, further experiments were performed. Mixing the free ligand with FeCl3 gave complex _A , which was subsequently reduced by hydrogenation:

Ligand, complex A and reduced material were analyzed by analytical LC (see Figure 6). Complex A was shown to have a longer retention time than the free ligand. However, after reduction, only peaks with the same retention time as the native ligand are shown. It must be concluded from this that the reduced complex is unstable, leading to decomplexation, which in turn yields uncomplexed ferrous iron and free ligand. Complementary experiments show corresponding results for Fe(II) oxidation in the presence of free ligand and air.

Example 4

To further investigate antibodies conjugated to iron complexes, a fluorescent (ie, Rhodamine 101)-labeled derivative of a lysine-based hydroxamic acid compound was synthesized (see Scheme 2).

Scheme 2. Synthesis of Rhodamine-Conjugated and OSu-Activated Hydroxamic Acid Compounds

This ligand was then conjugated to trastuzumab for further complexation with a non-OSu activating ligand also conjugated to Rhodamine 101 (see Figure 7).

The AB-conjugated complexes were purified by centrifugation in Vivaspin tubes (cutoff <30 kDa) by washing the conjugated complexes twice with citrate buffer (5 mL). The product was then diluted to 0.5 mL and fluorescence measurements were taken. As can be seen from Figure 8, AB-2 produced more fluorescence than unconjugated AB, and AB-1 produced less fluorescence than AB-2. Significant differences in the fluorescence of the two compounds were observed, indicating that the complexation with iron and the two auxiliary ligands was successful (see Figure 8, lower panel).

To examine whether the complex disintegrated by destabilization due to reduction of the ferric form of the complex to the ferrous complex, AB-2 was reduced to obtain AB-1. For this, AB-2 was reduced for 1 hour at RT under a hydrogen atmosphere and purified by Vivaspin centrifugation and washed twice with 5 mL of citrate buffer. Fluorescence measurements were then carried out (see Figure 9), from which it was observed that the control subjected to the same procedure (except hydrogenation) again showed -20% more fluorescence than the reduced product. This indicates that AB-2 is reduced to give AB-1.

Example 5

To assess whether an antibody conjugate containing a payload bound to an antibody via ferric iron is a) actually endocytosed into cells expressing the appropriate antigen, and b) remains sufficiently stable near the cell membrane in physiological media, Trastuzumab conjugates were prepared as described in Scheme 3. As the first linker, a lysine-based hydroxamic acid compound also conjugated to BODIPY was used. The hydroxamic acid portion of this structure enables the complexation of ferric iron with two auxiliary ligands. As ancillary ligands, again a lysine-based hydroxamic acid compound was used, in this case conjugated to rhodamine 101. Thus, a conjugate of trastuzumab containing a covalently linked BODIPY label, capable of complexing ferric iron, and two rhodamine-containing ligands, which are non-covalently bound to the antibody via the ferric iron complex, were generated. Valence conjugation.

SK-BR3- cells were grown overnight on coverslips with Polyornithin coating. These cells were then incubated with GVFe66b for up to 30 minutes or 6 hours, washed twice with PBS buffer and fixed (PFA).

Fluorescence microscopy after 30 minutes and 6 hours showed BODIPY and rhodamine signals on the cell surface after 1 hour and in the endosomal compartment after 6 hours. From this observation it must be concluded that the conjugate retains some stability until internalization.

Citrate buffer

Protocol 3. Preparation of trastuzumab-conjugated ADCs using the ferric linker concept. Compound GVFe49 was prepared by standard literature based synthesis. Subsequently, GVFe49 was conjugated with commercially available BODIPY-NHS to give compound GVFe60, which was followed by deprotection to give GVFe61 and -OSu activation to give GVFe62. This compound is next used for conjugation with antibodies. Subsequently, the conjugate was incubated with FeCl3 and compound _GVFe54 , which was synthesized by the same method as described for compound GVFe49.

Claims (24)

1. antibody-drug conjugates, it includes antibody, at least one ferric iron and at least one and ferric iron in conjunction with antibody In conjunction with drug molecule.

2. the antibody-drug conjugates of claim 1, wherein when antibody-drug conjugates in blood when, drug molecule not from Antibody-drug conjugates release.

3. the antibody-drug conjugates of claims 1 or 2, wherein when antibody-drug conjugates are in the inner body compartment of cell When, drug molecule is discharged from antibody-drug conjugates.

4. antibody-drug conjugates described in any one of claims 1 to 3, wherein at least two drug molecule and one three Valence iron combines.

5. the antibody-drug conjugates of any one of the claims, wherein in the conjugate each antibody drug molecule Average is at least 10.

6. the antibody-drug conjugates of any one of the claims, wherein drug molecule is anticancer agent.

7. the antibody-drug conjugates of any one of the claims, wherein antibody can be anti-in conjunction with what is expressed on tumour cell It is former.

8. the antibody-drug conjugates of any one of the claims, wherein drug molecule includes that ferric iron is combined to be complexed Part.

9. the antibody-drug conjugates of any one of claim 1 to 7, wherein drug molecule is coupled in conjunction with ferric iron network Close part.

10. the antibody-drug conjugates of claim 8 or 9, wherein the iron complexing moiety is selected from hydroxamic acid groups, catechu Phenol moieties and carboxylic moiety.

11. the antibody-drug conjugates of any one of the claims, have the following structure:

Ab[-L1-Me(-L2-D)_n]_m,

Wherein

Ab is antibody,

L1 is the first connector,

Me is ferric iron,

L2 is the second connector or is not present,

D is drug molecule,

M range is 1 to 10, and

N range is 1 to 3.

12. the antibody-drug conjugates of any one of the claims, wherein if ferric iron is reduced, drug molecule from It is discharged in ferric iron.

13. the antibody-drug conjugates of any one of the claims, wherein drug molecule is in the pH less than 5 from ferric iron Release.

14. the antibody-drug conjugates of any one of the claims are stablized in pH8.

15. the antibody-drug conjugates of any one of the claims are stablized in pH5.

16. pharmaceutical composition, it includes the antibody-drug conjugates of any one of the claims.

17. the antibody-drug conjugates of any one of claim 1-15 or the pharmaceutical composition of claim 16, are used to treat Method.

18. the antibody-drug conjugates of any one of claim 1-15 or the pharmaceutical composition of claim 16, are used to treat Cancer.

19. the purposes that ferric iron drug molecule in preventing or reducing blood is discharged from antibody-drug conjugates, wherein trivalent Iron is complexed to form antibody-drug conjugates by antibody and drug molecule.

20. the purposes of claim 18, wherein drug molecule is released from antibody-drug conjugates in the inner body compartment of cell It puts.

21. the purposes of claim 19 or 20, wherein antibody-drug conjugates are as determined in any one of claim 1 to 15 The antibody-drug conjugates of justice.

22. the antibody-drug conjugates of any one of claim 1-15 in preventing or reducing blood drug molecule from antibody-medicine The purposes discharged in object conjugate.

23. preventing or reducing the method that drug molecule in blood is discharged from antibody-drug conjugates, this method includes by trivalent Iron and the complexing of antibody and drug molecule are to form antibody-drug conjugates.

24. the method for claim 23, wherein antibody-drug conjugates are as defined in any one of claim 1 to 15 Antibody-drug conjugates.